The present invention is directed to a lipid based delivery vehicle. In particular, this invention is directed at cytoplasmic and/or nuclear delivery vehicles comprising a non-cationic lipid, a fusogenic peptide and a substance for delivery.
Advances in cellular and molecular biology, particularly the identification of defective genes, has increased the need for efficient vehicles to deliver substances such as genes, antisense molecules, ribozymes, and various regulators and therapeutics both intracellularly and intranuclearly.
To date, delivery vehicles have included replication-defective adenoviral vectors, cationic liposomes and protein-cationic peptides. For example, one study reports a system to deliver DNA in vitro by covalently attaching the surfactant associated protein B (SP-B) to a 10 kDa polylysine. See, Baatz, J., et al., PNAS USA, 91:2547-2551 (1994). However, this study also reports that the transfection of cells is performed in serum-free medium and is most effective with the addition of adenovirus. Other studies report on the use of the N-terminal 25 amino acids of the SP-B for use in lipid mixing, but do not report on substance delivery or use with cell membranes. See, e.g., Longmuir, et al., 1992 ASBMB/Biophysical Society abstract; Longmuir, et al., 1993 Biophysical Society abstract.
It is therefore an object of the present invention to provide a delivery vehicle comprising a non-cationic lipid for cytoplasmic and/or nuclear delivery of substances wherein the vehicle is stable and can be used in biological extracellular fluids typically found in animals, particularly blood serum.
It is also an object of the present invention to provide a delivery vehicle for cytoplasmic and/or nuclear delivery comprising a fusogenic peptide. It is particularly an object to provide functional portions of fusogenic peptides, and variants thereof which are characterized by their ability to facilitate fusion between a cell membrane and a liposome comprising the fusogenic peptide. It is also an object to provide fusogenic peptides which are characterized by their ability to facilitate transfer of substances across cellular membranes. It is also an object to provide variants of fusogenic peptides which exhibit lower cytotoxicity than the corresponding native fusogenic peptide.
It is also an object of the present invention to provide methods for assembling the aforementioned delivery vehicles as well as to provide the components used therein. It is further an object to provide methods for use of said delivery vehicles.
In accordance with the foregoing objectives, provided herein are liposome complexes and the components thereof for use in cytoplasmic and/or nuclear delivery of substances. Methods of making and using such liposome complexes are also provided. In one of the embodiments, components of the liposome complex include a non-cationic lipid and a fusogenic peptide. In some embodiments, the liposome complex further includes a substance to be delivered to the cytoplasm and/or nucleus.
The fusogenic peptides of the invention are characterized by their ability to facilitate fusion between a cell membrane and a liposome comprising the fusogenic peptide. Such peptides are also characterized by their ability to facilitate transfer of substances across cell membranes, particularly eukaryotic cell membranes. In a preferred embodiment, the fusogenic peptide comprises the consensus sequence: X-H-XXHHX-H-X-H (SEQ ID NO: 7).
In the consensus sequence, SEQ ID NO:7, xe2x80x9cXxe2x80x9d is any naturally occuring or synthetic hydrophobic amino acid. Examples of hydrophobic amino acids include alanine (Ala), valine (Val), isoleucine (Ile), leucine (Leu), norleucine (NorLeu), and phenylalanine (Phe). It is understood that variant or modified amino acids which retain or are altered to have hydrophobicity can be utilized. Further in the consensus sequence, at least 3 of the xe2x80x9cHxe2x80x9d positions are occupied by any naturally occuring or synthetic amino acid having a pKa of about 5 to about 7, and preferably about 6, wherein any remaining xe2x80x9cHxe2x80x9d positions are occupied by any amino acid, preferably a hydrophilic amino acid. Examples of amino acids having a suitable pKa include histidine, 1-methyl-histidine and 3-methyl histidine. Still further in the consensus sequence, xe2x80x9c-xe2x80x9d is any amino acid. Other embodiments include peptides derived from the pulmonary surfactant protein B (SP-B).
The non-cationic lipids provided herein are characterized by their ability to complex stably with fusogenic peptides and substance(s) to be delivered across cell membranes in the presence of biological fluids such as blood serum. Embodiments of such non-cationic lipids include 1,2-dimyristoleoyl--sn-glycero-3-phosphocholine (14:1-PC), and 1,2-dilauroyl-sn-glycero-3-phosphocholine (12:0-PC).
Any substance which can potentially modify the genotype or phenotype of the cell can be delivered according to the invention. Examples of substances to be delivered include genes, RNA, oligonucleotides, antisense molecules, ribozymes, peptides, factors and various regulators and therapeutics. In a preferred embodiment, when the substance is to be delivered to the inside of the nucleus, a nuclear localization signal peptide is also included.
The fusogenic peptides of the invention at physiological pH generally have a net positive charge. However, in some of the embodiments provided herein, the fusogenic peptide does not contain multiple positive charges at neutral pH and above. In such embodiments, and when the substance to be delivered is a negatively charged polymer such as DNA or RNA, additional components are preferably used in assembling the liposome complex. For example, in one embodiment, a positively charged companion peptide such as a nuclear localization signal peptide (NLSP) reacted with N-iodoacetyl-(1,2-dioleoyl)-sn-glycero-3-phosphoethanolamine (N-iodoacetyl-DOPE) or N-(3-maleimido)benzoyl-DOPE (DOPE-MBS) is additionally utilized.